Study on low-temperature lipase of psychrophilic bacterium 2-5-10-1 isolated from deep sea of Southern Ocean

A strain of psychrophilic bacterium, 2-5-10-1, which produces low-temperature lipase, is isolated from the deep sea of Prydz Bay in Southern Ocean. The highest lipase secretion of this strain is observed at 5 degreesC and this temperature is also for optimal growth. Tween 80 and olive oil enhance secretion of lipase. The optimal temperature and pH for lipase activity are 35 degreesC and 7.5 degreesC respectively. At 0degreesC, the lipase still has 37% relative enzyme activity. The lipase shows high thermolability, more than 50% activity lost after incubation at 60 degreesC for 15 min. EDTA has no effect on lipase activity, indicating the lipase activity is independent of divalent cation. In contrast, the lipase activity is inhibited drastically by Cu2+ and Zn2+.

Structure and function of a novel cold regulated cold shock domain containing protein from an obligate psychrophilic yeast,Glaciozyma antarctica

Cold shock domain(CSD)-containing proteins are one of the groups of the evolutionarily conserved nucleic acid-binding proteins in all three domains of life consisting of an ancient beta-barrel fold that serves to bind nucleic acids.The c DNA of a novel protein-coding gene containing CSD was cloned from Glaciozyma antarctica designated as Ga16676.The full length of Ga16676 gene with the size of 1335 bp encodes for an N-terminal CSD with conserved nucleic acids binding motif RNP1 and RNP2.The Ga16676 gene was cloned in p ET30 Ek/LIC,sequenced,expressed and its resistance towards cold was characterized.Recombinant protein expression of Ga16676 showed overexpressed soluble expression in both supernatant and pellet forms at 20℃.The effects of recombinant CSD protein overexpression on colony formation shows that E.coli cells were able to grow at 37℃and 20℃but not at 4℃while E.coli_Ga16676 cells were able to grow at all temperatures tested.In addition,E.coli_Ga16676 cells showed higher growth rate compared to empty E.coli cells at 10℃.Structural analysis of Ga16676 reveals some interesting findings such as more aromatic interactions for efficient binding in low energy environment,a longer loop that may contribute to structural flexibility and clustering of charged amino acids on the protein surface that is important for protein stability and flexibility.

Isolation of novel psychrophilic bacteria from Arctic sea ice

The phylogenetic diversity of cuhurable psychrophilic bacteria associated with sea ice from the high latitude regions of Canadian Basin and Chukchi Sea, Arctic, was investigated. A total of 34 psychropilic strains were isolated using three methods of （ Ⅰ ） dilution plating （ at 4 ℃ ）, （ Ⅱ ） bath culturing （ at - 1 ℃ ） and dilution plating, and （ Ⅲ） cold shock （ -20 ℃ for 24 h）, bath culturing and dilution plating under aerobic conditions. Sea-ice samples were exposed to -20 ℃ for 24 h that might reduce the number of common microorganisms and encourage outgrowth of psyehrophilic strains. This process might be able to be introduced to isolation psychrophilic bacteria from other environmental samples in future study. 16S rDNA nearly full-length sequence analysis revealed that psychrophilic strains felled in two phylo- genetic divisions, γ-proteobacteria （in the genera ColweUia,Marinobaeter、Shewanella,Glaciecola,Marinomortas and Pseudoalteromortas ） and Cytophaga-Flexibacter-Baeteroides （ Flavobacterlum and Psyehroflexus）. Fifteen of bacterial isolates quite likely represented novel species （16S rDNA sequence similarity below 98% ）. One of strains （BSi20002） from Canadian Basin showed 100% sequence similarity to that of Marinobacter sp. ANT8277 isolated from the Antarctic Weddell sea ice, suggesting bacteria may have a bipolar distribution at the species level.

Isolation and Characterization of Psychrophilic Algae in Boron Enriched Pond on Boron Deposit of Qinghai

1 Introduction Cold environments on the planet occur in the Antarctic,the Arctic and high mountains which cover with ice and snow permanently.Microorganism adapted to cold habitats has been detected,but most of them were prokaryotes.(Morgan-Kiss et al,2006;jungbut et al,2012).Research on cold-adapted microorganism has

Transposon mutagenesis of Psychrobacter cryohalolentis PAMC 21807 by tri-parental conjugation

Random mutagenesis is commonly used to study gene function. The screening of mutants exhibiting specific pheno- types assists in the identification of phenotype-related genes. In the current study, we isolated Antarctic bacteria, and developed a transposon Tn5 mutagenesis system. A total of 26 strains were isolated from seawater and freshwater near Antarctic King Sejong Research Station, King George Island. Six Psychrobacter strains were identified as psychrophilic, with optimal growth tempera- tures of 10~C or 15~C Psychrobacter cryohalolentis PAMC 21807 with a high growth rate at 4~C was selected for transposon mutagenesis. Tri-parental conjugation with a plasmid containing Tn5 produced 13 putative recombinants containing the selectable marker. Genomic Southern hybridization confirmed Tn5 existed as episomes for seven recombinants, and for a single recombinant, Tn5 was integrated into the genome of Psychrobacter cryohalolentis PAMC 21807. The result indicates that the mutagenesis method, although successful, has a relatively low rate. The psychrophilic bacteria isolated in this study may be a useful resource for studying cold adaptation mechanisms, and the mutagenesis method can be applied to genetic analysis.

Heterologous Expression of Thermolabile Proteins Enhances Thermotolerance in Escherichia coli

Heat shock proteins (HSPs) play important roles in the mechanism of cellular protection against various environmental stresses. It is well known that accumulation of misfolded proteins in a cell triggers the HSPs expression in prokaryotes as well as eukaryotes. In this study, we heterologously expressed two proteins in E. coli, namely, citrate synthase (CpCSY) and malate dehydrogenase (CpMDH) from a psychrophilic bacterium Colwellia psychrerythraea 34H (optimal growth temperature 8°C). Our analyses using circular dichromism along with temperature-dependant enzyme activities measured in purified or direct cell extracts confirmed that the CpCSY and CpMDH are thermolabile and present in misfolded form even at physiological growth temperature. We observed that the cellular levels of HSPs, both GroEL and DnaK cheperonins were increased. Similarly, higher levels were observed for sigma factor s32 which is specific to heat-shock protein expression. These results suggest that the misfolded-thermolabile proteins expressed in E. coli induced the heat shock response. Furthermore, heat treatment (53°C) to wild type E. coli noticeably delayed their growth recovery but cells expressing CpCSY and CpMDH recovered their growth much faster than that of wild type E. coli. This reveals that the HSPs expressed in response to misfolded-thermolabile proteins protected E. coli against heat-induced damage. This novel approach may be a useful tool for investigating stress-tolerance mechanisms of E. coli.

Gene duplication and functional divergence of new genes contributed to the polar acclimation of Antarctic green algae

Psychrophilic microalgae successfully survive in the extreme and highly variable polar ecosystems,which represent the energy base of most food webs and play a fundamental role in nutrient cycling.The success of microalgae is rooted in their adaptive evolution.Revealing how they have evolved to thrive in extreme polar environments will help us better understand the origin of life in polar ecosystems.We isolated a psychrophilic unicellular green alga,Microglena sp.YARC,from Antarctic sea ice which has a huge genome.Therefore,we predicted that gene replication may play an important role in its polar adaptive evolution.We found that its protein-coding gene number significantly increased and the duplication time was dated between 37 and 48 million years ago,which is consistent with the formation of the circumpolar Southern Ocean.Most duplicated paralogous genes were enriched in pathways related to photosynthesis,DNA repair,and fatty acid metabolism.Moreover,there were a total of 657 Microglena-specific families,including collagen-like proteins.The divergence in the expression patterns of the duplicated and species-specific genes reflects sub-and neo-functionalization during stress acclimation.Overall,key findings from this study provide new information on how gene duplication and their functional novelty contributed to polar algae adaptation to the highly variable polar environmental conditions.

Performance of low temperature Microbial Fuel Cells(MFCs)catalyzed by mixed bacterial consortia

Microbial Fuel Cells（MFCs） are a promising technology for treating wastewater in a sustainable manner. In potential applications, low temperatures substantially reduce MFC performance. To better understand the effect of temperature and particularly how bioanodes respond to changes in temperature, we investigated the current generation of mixed-culture and pure-culture MFCs at two low temperatures, 10°C and 5°C. The results implied that the mixed-culture MFC sustainably performed better than the pure-culture（Shewanella） MFC at 10°C, but the electrogenic activity of anodic bacteria was substantially reduced at the lower temperature of 5°C. At 10°C, the maximum output voltage generated with the mixed-culture was 540–560 m V, which was 10%–15% higher than that of Shewanella MFCs. The maximum power density reached 465.3 ± 5.8 m W/m^2 for the mixed-culture at10°C, while only 68.7 ± 3.7 m W/m^2 was achieved with the pure-culture. It was shown that the anodic biofilm of the mixed-culture MFC had a lower overpotential and resistance than the pure-culture MFC. Phylogenetic analysis disclosed the prevalence of Geobacter and Pseudomonas rather than Shewanella in the mixed-culture anodic biofilm, which mitigated the increase of resistance or overpotential at low temperatures.

Effect of the chlortetracycline addition method on methane production from the anaerobic digestion of swine wastewater

Effects of antibiotic residues on methane production in anaerobic digestion are commonly studied using the following two antibiotic addition methods：（1） adding manure from animals that consume a diet containing antibiotics, and（2） adding antibiotic-free animal manure spiked with antibiotics. This study used chlortetracycline（CTC） as a model antibiotic to examine the effects of the antibiotic addition method on methane production in anaerobic digestion under two different swine wastewater concentrations（0.55 and 0.22 mg CTC/g dry manure）. The results showed that CTC degradation rate in which manure was directly added at 0.55 mg CTC/g（HSPIKE treatment） was lower than the control values and the rest of the treatment groups. Methane production from the HSPIKEtreatment was reduced（p 〈 0.05） by 12% during the whole experimental period and 15% during the first 7 days. The treatments had no significant effect on the pH and chemical oxygen demand value of the digesters, and the total nitrogen of the0.55 mg CTC/kg manure collected from mediated swine was significantly higher than the other values. Therefore, different methane production under different antibiotic addition methods might be explained by the microbial activity and the concentrations of antibiotic intermediate products and metabolites. Because the primary entry route of veterinary antibiotics into an anaerobic digester is by contaminated animal manure, the most appropriate method for studying antibiotic residue effects on methane production may be using manure from animals that are given a particular antibiotic, rather than adding the antibiotic directly to the anaerobic digester.